Molecular basis for ligation of mismatched DNA ends. The nonhomologous end joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) produced by ionizing radiation or by enzymatic cleavage to generate immunological diversity. To ensure chromosomal integrity, NHEJ must join the ends while optimizing the preservation of DNA sequence. This proposal focuses on an unprecedented ligase activity that can join DNA ends with mismatched overhangs. This mismatched end (MEnd) ligase activity requires Ku, XRCC4/Ligase IV and Cernunnos. By ligating ends refractory to all other ligases, MEnd ligase optimizes preservation of DNA sequence. What is the molecular basis for this remarkable activity? Crystal structures and mutant forms of Cernunnos and XRCC4 led to a surprising hypothesis: Cernunnos and XRCC4 form a filament that aligns mismatched DNA ends for ligation. Powerful methods will be used to either support the hypothesis or generate an alternative model. The specific aims are to: Aim 1: Characterize protein interactions in the MEnd ligase complex. Misincorporation proton-alkyl exchange (MPAX) will be applied to the MEnd ligase proteins. MPAX randomly misincorporates cysteines into the targeted protein, and then identifies exposed cysteine residues by alkylation. This will permit high-throughput mapping of exposed residues that are buried by protein-protein interactions. Aim 2: Determine the effect of mutations on NHEJ. Mutant proteins will be synthesized and tested for MEnd ligase activity, NHEJ in extracts and V(D)J recombination in cells. Clinical mutations in Cernunnos and Ligase IV confer cellular radiosensitivity. However, some of the mutations cause microcephaly and growth delay, while others cause severe combined immunodeficiency. We will determine if such mutations produce distinct biochemical abnormalities in MEnd ligase. Aim 3: Analyze the assembly of the MEnd ligase complex on DNA ends. To probe alignment of DNA ends by MEnd ligase, we will test various mismatched overhangs. EMSAs will determine whether Ku stabilizes binding of Cernunnos and XL to DNA. Photo-cross-linking will determine the positions of proteins at the DNA ends. Pull-down assays will test whether MEnd ligase facilitates synapsis of ends. Preliminary electron microscopy images suggest spontaneous formation of XRCC4/Cernunnos filament- like structures, and we will determine whether these structures are the hypothesized filament. Our studies have implications for human health. Mutations in the Ligase IV and Cernunnos genes produce inherited diseases of immune deficiency and cancer susceptibility. Environmental DSBs can cause cancer. Many effective anticancer agents generate DSBs. Therefore, understanding the molecular basis for repairing DSBs may lead to improvements in cancer prevention and treatment. PUBLIC HEALTH RELEVANCE: This proposal aims to understand nonhomologous end-joining, a pathway that rejoins broken chromosomes. Such breaks occur after radiation or during the process that produces immunity against infection. The core reaction for this pathway can join damaged DNA ends, even when conventional enzymes cannot. Defects in the core reaction lead to immune deficiency, growth retardation, and cancer.